Elicitation of Both Anti HIV-1 Env Humoral and Cellular Immunities by Replicating Vaccinia Prime Sendai Virus Boost Regimen and Boosting by CD40Lm

For protection from HIV-1 infection, a vaccine should elicit both humoral and cell-mediated immune responses. A novel vaccine regimen and adjuvant that induce high levels of HIV-1 Env-specific T cell and antibody (Ab) responses was developed in this study. The prime-boost regimen that used combinations of replication-competent vaccinia LC16m8Δ (m8Δ) and Sendai virus (SeV) vectors expressing HIV-1 Env efficiently produced both Env-specific CD8⁺ T cells and anti-Env antibodies, including neutralizing antibodies (nAbs). These results sharply contrast with vaccine regimens that prime with an Env expressing plasmid and boost with the m8Δ or SeV vector that mainly elicited cellular immunities. Moreover, co-priming with combinations of m8Δs expressing Env or a membrane-bound human CD40 ligand mutant (CD40Lm) enhanced Env-specific CD8⁺ T cell production, but not anti-Env antibody production. In contrast, priming with an m8Δ that coexpresses CD40Lm and Env elicited more anti-Env Abs with higher avidity, but did not promote T cell responses. These results suggest that the m8Δ prime/SeV boost regimen in conjunction with CD40Lm expression could be used as an immunization platform for driving both potent cellular and humoral immunities against pathogens such as HIV-1.     

Utility of human immunodeficiency virus type 1 envelope as a T-cell immunogen

Human immunodeficiency virus (HIV)-specific CD8 T lymphocytes are important for the control of viremia, but the relative utility of responses to the various HIV proteins is controversial. Immune responses that force escape mutations that exact a significant fitness cost from the mutating virus would help slow progression to AIDS. The HIV envelope (Env) protein is subject to both humoral and cellular immune responses, suggesting that multiple rounds of mutation are needed to facilitate viral escape. The Gag protein, however, has recently been shown to elicit a more effective CD8 T-cell immune response in humans. We studied 30 pigtail macaques for their CD8 T-lymphocyte responses to HIV-1 Env and simian immunodeficiency virus (SIV) Gag following prime/boost vaccination and intrarectal challenge with simian-human immunodeficiency virus SHIV_mn229. Eight CD8 Env-specific T-cell epitopes were identified and mapped in 10 animals. Animals that generated Env-specific CD8 T-cell responses had equivalent viral loads and only a modest advantage in retention of peripheral CD4 T lymphocytes compared to those animals without responses to Env. This contrasts with animals that generated CD8 T-cell responses to SIV Gag in the same trial, demonstrating superior control of viral load and a larger advantage in retention of peripheral CD4 T cells than Gag nonresponders. Mutational escape was common in Env but, in contrast to mutations in Gag, did not result in the rapid emergence of dominant escape motifs, suggesting modest selective pressure from Env-specific T cells. These results suggest that Env may have limited utility as a CD8 T-cell immunogen.     

Induction of broad cross-subtype-specific HIV-1 immune responses by a novel multivalent HIV-1 peptide vaccine in cynomolgus macaques

One of the major obstacles in the design of an effective vaccine against HIV-1 is its antigenic variation, which results in viral escape from the immune system. Through a bioinformatics approach, we developed an innovative multivalent HIV-1 vaccine comprised of a pool of 176 lipidated and nonlipidated peptides representing variable regions of Env and Gag proteins. The potency and breadth of the candidate vaccine against a panel of HIV-1 subtypes was evaluated in nonhuman primate (cynomolgus macaques) and humanized mouse (HLA-A2.1) models. The results demonstrate strong immunogenicity with both breadth (humoral and cellular immunity) and depth (immune recognition of widely divergent viral sequences) against heterologous HIV-1 subtypes A-F.     

Altering an artificial Gagpolnef polyprotein and mode of ENV co-administration affects the immunogenicity of a clade C HIV DNA vaccine

HIV-1 candidate vaccines expressing an artificial polyprotein comprising Gag, Pol and Nef (GPN) and a secreted envelope protein (Env) were shown in recent Phase I/II clinical trials to induce high levels of polyfunctional T cell responses; however, Env-specific responses clearly exceeded those against Gag. Here, we assess the impact of the GPN immunogen design and variations in the formulation and vaccination regimen of a combined GPN/Env DNA vaccine on the T cell responses against the various HIV proteins. Subtle modifications were introduced into the GPN gene to increase Gag expression, modify the expression ratio of Gag to PolNef and support budding of virus-like particles. I.m. administration of the various DNA constructs into BALB/c mice resulted in an up to 10-fold increase in Gag- and Pol-specific IFNγ(+) CD8(+) T cells compared to GPN. Co-administering Env with Gag or GPN derivatives largely abrogated Gag-specific responses. Alterations in the molar ratio of the DNA vaccines and spatially or temporally separated administration induced more balanced T cell responses. Whereas forced co-expression of Gag and Env from one plasmid induced predominantly Env-specific T cells responses, deletion of the only H-2(d) T cell epitope in Env allowed increased levels of Gag-specific T cells, suggesting competition at an epitope level. Our data demonstrate that the biochemical properties of an artificial polyprotein clearly influence the levels of antigen-specific T cells, and variations in formulation and schedule can overcome competition for the induction of these responses. These results are guiding the design of ongoing pre-clinical and clinical trials.     

Immunogenicity of multiple gene and clade human immunodeficiency virus type 1 DNA vaccines

The ability to elicit an immune response to a spectrum of human immunodeficiency virus type 1 (HIV-1) gene products from divergent strains is a desirable feature of an AIDS vaccine. In this study, we examined combinations of plasmids expressing multiple HIV-1 genes from different clades for their ability to elicit humoral and cellular immune responses in mice. Immunization with a modified Env, gp145DeltaCFI, in combination with a Gag-Pol-Nef fusion protein plasmid elicited similar CD4(+) and CD8(+) cellular responses to immunization with either vector alone. Further, when mice were immunized with a mixture of Env from three clades, A, B, and C, together with Gag-Pol-Nef, the overall potency and balance of CD4(+)- and CD8(+)-T-cell responses to all viral antigens were similar, with only minor differences noted. In addition, plasmid mixtures elicited antibody responses comparable to those from individual inoculations. These findings suggest that a multigene and multiclade vaccine, including components from A, B, and C Env and Gag-Pol-Nef, can broaden antiviral immune responses without immune interference. Such combinations of immunogens may help to address concerns about viral genetic diversity for a prospective HIV-1 vaccine.     

HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees: A Phase I Randomized Trial

Background

We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers.

Methods

HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA.

Results

The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4⁺ and/or CD8⁺ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4⁺ and CD8⁺ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8⁺ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients.

Conclusion

Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use.

Trial Registration

International Standard Randomised Controlled Trial Number (ISRCTN) 60284968     

Identification of cross-clade CTL epitopes in HIV-1 clade A/E-infected individuals by using the clade B overlapping peptides

Identification of cross-clade T cell epitopes is one of key factors for the development of a widely applicable AIDS vaccine. We here investigated cross-clade CD8⁺ T cell responses between clade B and A/E viruses in chronically HIV-1 clade A/E-infected Japanese individuals. CD8⁺ T cell responses to 11-mer overlapping peptides derived from Nef, Gag, and Pol clade B consensus sequences were at a similar level to those to the same peptides found in clade B-infected individuals. Fifteen cross-clade CTL epitopes were identified from 13 regions where the frequency of responders was high in the clade A/E-infected individuals. The sequences of 6 epitopes were conserved between the clade B and clade A/E viruses whereas 9 epitopes had different amino acid sequences between the 2 viruses. CD8⁺ T cells specific for the 6 conserved epitopes recognized cells infected with the clade A/E virus, whereas those for 8 diverse epitopes recognized both the clade A/E virus-infected and clade B-infected cells. All of the cross-clade CD8⁺ T cells specific for conserved and diverse epitopes were detected in chronically HIV-1 clade A/E-infected individuals. These results show that in addition to conserved regions polymorphic ones across the clades can be targets for cross-clade CTLs.     

Vaccination with a Fusion Protein That Introduces HIV-1 Gag Antigen into a Multitrimer CD40L Construct Results in Enhanced CD8+ T Cell Responses and Protection from Viral Challenge by Vaccinia-Gag

CD40 ligand (CD40L, CD154) is a membrane protein that is important for the activation of dendritic cells (DCs) and DC-induced CD8⁺ T cell responses. To be active, CD40L must cluster CD40 receptors on responding cells. To produce a soluble form of CD40L that clusters CD40 receptors necessitates the use of a multitrimer construct. With this in mind, a tripartite fusion protein was made from surfactant protein D (SPD), HIV-1 Gag as a test antigen, and CD40L, where SPD serves as a scaffold for the multitrimer protein complex. This SPD-Gag-CD40L protein activated CD40-bearing cells and bone marrow-derived DCs in vitro. Compared to a plasmid for Gag antigen alone (pGag), DNA vaccination of mice with pSPD-Gag-CD40L induced an increased number of Gag-specific CD8⁺ T cells with increased avidity for major histocompatibility complex class I-restricted Gag peptide and improved vaccine-induced protection from challenge by vaccinia-Gag virus. The importance of the multitrimeric nature of the complex was shown using a plasmid lacking the N terminus of SPD that produced a single trimer fusion protein. This plasmid, pTrimer-Gag-CD40L, was only weakly active on CD40-bearing cells and did not elicit strong CD8⁺ T cell responses or improve protection from vaccinia-Gag challenge. An adenovirus 5 (Ad5) vaccine incorporating SPD-Gag-CD40L was much stronger than Ad5 expressing Gag alone (Ad5-Gag) and induced complete protection (i.e., sterilizing immunity) from vaccinia-Gag challenge. Overall, these results show the potential of a new vaccine design in which antigen is introduced into a construct that expresses a multitrimer soluble form of CD40L, leading to strongly protective CD8⁺ T cell responses.     

Polymorphisms in HLA class I genes associated with both favorable prognosis of human immunodeficiency virus (HIV) type 1 infection and positive cytotoxic T-lymphocyte responses to ALVAC-HIV recombinant canarypox vaccines

Carriers of certain human leukocyte antigen class I alleles show favorable prognosis of human immunodeficiency virus type 1 (HIV-1) infection, presumably due to effective CD8(+) cytotoxic T-lymphocyte responses, but close relationships between class I variants mediating such responses to natural and to vaccine HIV-1 antigen have not been established. During 6 to 30 months of administration and follow-up in trials of ALVAC-HIV recombinant canarypox vaccines, cells from 42% of 291 HIV-1-negative vaccinated subjects typed at class I loci responded to an HIV-1 protein in a lytic bulk CD8(+) cytotoxic T-lymphocyte assay. By 2 weeks after the second dose, higher proportions of vaccinees carrying one of two alleles consistently associated with slower progression of natural HIV-1 infection reacted at least once: B*27 carriers reacted to Gag (64%; odds ratio [OR] = 10.3, P = 0.001) and Env (36%; OR = 4.6, P = 0.04), and B*57 carriers reacted to Env (44%; OR = 6.6, P < 0.05). By 2 weeks after the third or fourth dose, B*27 carriers had responded (two or more reactions) to Gag (33%; OR = 4.4, P < 0.05) and B*57 carriers had responded to both Gag (39%; OR = 5.3, P = 0.013) and Env (39%; OR = 9.5, P = 0.002). Homozygosity at class I loci, although conferring an unfavorable prognosis following natural infection, showed no such disadvantage for vaccine response. Individual class I alleles have not previously demonstrated such clear and consistent relationship with both the clinical course of an infection and cellular immunity to a vaccine against the infectious agent. This proof of principle that class I an alleles modulate both processes has implications for development of HIV-1 and presumably other vaccines.     

Broad and Cross-Clade CD4+ T-Cell Responses Elicited by a DNA Vaccine Encoding Highly Conserved and Promiscuous HIV-1 M-Group Consensus Peptides

T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4⁺ T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4⁺ T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4⁺ T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IA^b and IA^d. Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-γ secretion against 11 out of the 27 peptides in BALB/c mice; CD4⁺ and CD8⁺ T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4⁺ and CD8⁺ T cells, able to simultaneously proliferate and produce IFN-γ and TNF-α, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS.     

Improving Adaptive and Memory Immune Responses of an HIV/AIDS Vaccine Candidate MVA-B by Deletion of Vaccinia Virus Genes (C6L and K7R) Blocking Interferon Signaling Pathways

Poxvirus vector Modified Vaccinia Virus Ankara (MVA) expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (termed MVA-B) is a promising HIV/AIDS vaccine candidate, as confirmed from results obtained in a prophylactic phase I clinical trial in humans. To improve the immunogenicity elicited by MVA-B, we have generated and characterized the innate immune sensing and the in vivo immunogenicity profile of a vector with a double deletion in two vaccinia virus (VACV) genes (C6L and K7R) coding for inhibitors of interferon (IFN) signaling pathways. The innate immune signals elicited by MVA-B deletion mutants (MVA-B ΔC6L and MVA-B ΔC6L/K7R) in human macrophages and monocyte-derived dendritic cells (moDCs) showed an up-regulation of the expression of IFN-β, IFN-α/β-inducible genes, TNF-α, and other cytokines and chemokines. A DNA prime/MVA boost immunization protocol in mice revealed that these MVA-B deletion mutants were able to improve the magnitude and quality of HIV-1-specific CD4⁺ and CD8⁺ T cell adaptive and memory immune responses, which were mostly mediated by CD8⁺ T cells of an effector phenotype, with MVA-B ΔC6L/K7R being the most immunogenic virus recombinant. CD4⁺ T cell responses were mainly directed against Env, while GPN-specific CD8⁺ T cell responses were induced preferentially by the MVA-B deletion mutants. Furthermore, antibody levels to Env in the memory phase were slightly enhanced by the MVA-B deletion mutants compared to the parental MVA-B. These findings revealed that double deletion of VACV genes that act blocking intracellularly the IFN signaling pathway confers an immunological benefit, inducing innate immune responses and increases in the magnitude, quality and durability of the HIV-1-specific T cell immune responses. Our observations highlighted the immunomodulatory role of the VACV genes C6L and K7R, and that targeting common pathways, like IRF3/IFN-β signaling, could be a general strategy to improve the immunogenicity of poxvirus-based vaccine candidates.     

Deletion of Specific Immune-Modulatory Genes from Modified Vaccinia Virus Ankara-Based HIV Vaccines Engenders Improved Immunogenicity in Rhesus Macaques

Modified vaccinia virus Ankara (MVA) is a safe, attenuated orthopoxvirus that is being developed as a vaccine vector but has demonstrated limited immunogenicity in several early-phase clinical trials. Our objective was to rationally improve the immunogenicity of MVA-based HIV/AIDS vaccines via the targeted deletion of specific poxvirus immune-modulatory genes. Vaccines expressing codon-optimized HIV subtype C consensus Env and Gag antigens were generated from MVA vector backbones that (i) harbor simultaneous deletions of four viral immune-modulatory genes, encoding an interleukin-18 (IL-18) binding protein, an IL-1β receptor, a dominant negative Toll/IL-1 signaling adapter, and CC-chemokine binding protein (MVAΔ4-HIV); (ii) harbor a deletion of an additional (fifth) viral gene, encoding uracil-DNA glycosylase (MVAΔ5-HIV); or (iii) represent the parental MVA backbone as a control (MVA-HIV). We performed head-to-head comparisons of the cellular and humoral immune responses that were elicited by these vectors during homologous prime-boost immunization regimens utilizing either high-dose (2 × 10⁸ PFU) or low-dose (1 × 10⁷ PFU) intramuscular immunization of rhesus macaques. At all time points, a majority of the HIV-specific T cell responses, elicited by all vectors, were directed against Env, rather than Gag, determinants, as previously observed with other vector systems. Both modified vectors elicited up to 6-fold-higher frequencies of HIV-specific CD8 and CD4 T cell responses and up to 25-fold-higher titers of Env (gp120)-specific binding (nonneutralizing) antibody responses that were relatively transient in nature. While the correlates of protection against HIV infection remain incompletely defined, our results indicate that the rational deletion of specific genes from MVA vectors can positively alter their cellular and humoral immunogenicity profiles in nonhuman primates.     

HIV-1 Gag多表位嵌合基因的构建及表达蛋白的抗体制备

旨在通过构建Gag的抗原多表位融合基因及在原核系统的高表达,为HIV诊断及可能的疫苗制备提供试验基础。选定HIV-1 Gag基因中3个片段包含较多抗原表位的区域,设计带有酶切位点的引物,用PCR的方法从HIV-1HXB2全基因扩增编码这3个片段的基因序列,通过质粒提取、酶切、测序方法鉴定基因片段的正确性,SDS-PAGE和Western blotting测定融合蛋白的表达,并免疫动物制备相应抗体。结果显示,构建的HIV-1 Gag多表位嵌合基因的原核表达质粒,酶切和测序结果表明基因序列正确,基因全长576bp。在大肠杆菌BL21(DE3)中高效表达的重组蛋白分子量为27kD,以包涵体的形式存在。纯化目的蛋白免疫家兔,制备多克隆抗体IgG。ELISA和免疫荧光方法检测显示制备的多克隆抗体能具有特异性反应。成功构建和高表达了HIV-1 Gag多表位融合蛋白,纯化蛋白制备的抗体与HIV-1Gag有特异性结合。为进一步研究HIV-1奠定了试验基础。     

Full-Length HIV-1 Immunogens Induce Greater Magnitude and Comparable Breadth of T Lymphocyte Responses to Conserved HIV-1 Regions Compared with Conserved-Region-Only HIV-1 Immunogens in Rhesus Monkeys

A global HIV-1 vaccine will likely need to induce immune responses against conserved HIV-1 regions to contend with the profound genetic diversity of HIV-1. Here we evaluated the capacity of immunogens consisting of only highly conserved HIV-1 sequences that are aimed at focusing cellular immune responses on these potentially critical regions. We assessed in rhesus monkeys the breadth and magnitude of T lymphocyte responses elicited by adenovirus vectors expressing either full-length HIV-1 Gag/Pol/Env immunogens or concatenated immunogens consisting of only highly conserved HIV-1 sequences. Surprisingly, we found that the full-length immunogens induced comparable breadth (P = 1.0) and greater magnitude (P = 0.01) of CD8⁺ T lymphocyte responses against conserved HIV-1 regions compared with the conserved-region-only immunogens. Moreover, the full-length immunogens induced a 5-fold increased total breadth of HIV-1-specific T lymphocyte responses compared with the conserved-region-only immunogens (P = 0.007). These results suggest that full-length HIV-1 immunogens elicit a substantially increased magnitude and breadth of cellular immune responses compared with conserved-region-only HIV-1 immunogens, including greater magnitude and comparable breadth of responses against conserved sequences.     

Heterologous Prime-Boost Regimens with a Recombinant Chimpanzee Adenoviral Vector and Adjuvanted F4 Protein Elicit Polyfunctional HIV-1-Specific T-Cell Responses in Macaques

HIV-1-specific CD4⁺ and CD8⁺ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4⁺ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8⁺ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN (‘A’), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 (‘P’), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4⁺ and CD8⁺ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4⁺ T cells. Approximately 50% of AdC7-GRN-induced memory CD8⁺ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4⁺ and CD8⁺ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4⁺ and CD8⁺ T-cell responses and antibodies in macaques. Besides, adenoviral vector priming modulated the cytokine-expression profile of the protein-induced CD4⁺ T cells. Each regimen induced HIV-1-specific T-cell responses in systemic/local tissues in mice. This suggests that prime-boost regimens combining adjuvanted protein and low-seroprevalent chimpanzee adenoviral vectors represent an attractive vaccination strategy for clinical evaluation.     

Comparison of Heterologous Prime-Boost Strategies against Human Immunodeficiency Virus Type 1 Gag Using Negative Stranded RNA Viruses

This study analyzed a heterologous prime-boost vaccine approach against HIV-1 using three different antigenically unrelated negative-stranded viruses (NSV) expressing HIV-1 Gag as vaccine vectors: rabies virus (RABV), vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV). We hypothesized that this approach would result in more robust cellular immune responses than those achieved with the use of any of the vaccines alone in a homologous prime-boost regimen. To this end, we primed BALB/c mice with each of the NSV-based vectors. Primed mice were rested for thirty-five days after which we administered a second immunization with the same or heterologous NSV-Gag viruses. The magnitude and quality of the Gag-specific CD8⁺ T cells in response to these vectors post boost were measured. In addition, we performed challenge experiments using vaccinia virus expressing HIV-1 Gag (VV-Gag) thirty-three days after the boost inoculation. Our results showed that the choice of the vaccine used for priming was important for the detected Gag-specific CD8⁺ T cell recall responses post boost and that NDV-Gag appeared to result in a more robust recall of CD8⁺ T cell responses independent of the prime vaccine used. However, the different prime-boost strategies were not distinct for the parameters studied in the challenge experiments using VV-Gag but did indicate some benefits compared to single immunizations. Taken together, our data show that NSV vectors can individually stimulate HIV-Gag specific CD8⁺ T cells that are effectively recalled by other NSV vectors in a heterologous prime-boost approach. These results provide evidence that RABV, VSV and NDV can be used in combination to develop vaccines needing prime-boost regimens to stimulate effective immune responses.     

CD8 and CD4 Epitope Predictions in RV144: No Strong Evidence of a T-Cell Driven Sieve Effect in HIV-1 Breakthrough Sequences from Trial Participants

The modest protection afforded by the RV144 vaccine offers an opportunity to evaluate its mechanisms of protection. Differences between HIV-1 breakthrough viruses from vaccine and placebo recipients can be attributed to the RV144 vaccine as this was a randomized and double-blinded trial. CD8 and CD4 T cell epitope repertoires were predicted in HIV-1 proteomes from 110 RV144 participants. Predicted Gag epitope repertoires were smaller in vaccine than in placebo recipients (p = 0.019). After comparing participant-derived epitopes to corresponding epitopes in the RV144 vaccine, the proportion of epitopes that could be matched differed depending on the protein conservation (only 36% of epitopes in Env vs 84–91% in Gag/Pol/Nef for CD8 predicted epitopes) or on vaccine insert subtype (55% against CRF01_AE vs 7% against subtype B). To compare predicted epitopes to the vaccine, we analyzed predicted binding affinity and evolutionary distance measurements. Comparisons between the vaccine and placebo arm did not reveal robust evidence for a T cell driven sieve effect, although some differences were noted in Env-V2 (0.022≤p-value≤0.231). The paucity of CD8 T cell responses identified following RV144 vaccination, with no evidence for V2 specificity, considered together both with the association of decreased infection risk in RV 144 participants with V-specific antibody responses and a V2 sieve effect, lead us to hypothesize that this sieve effect was not T cell specific. Overall, our results did not reveal a strong differential impact of vaccine-induced T cell responses among breakthrough infections in RV144 participants.     

HIV-1 Conserved Elements p24CE DNA Vaccine Induces Humoral Immune Responses with Broad Epitope Recognition in Macaques

To target immune responses towards invariable regions of the virus, we engineered DNA-based immunogens encoding conserved elements (CE) of HIV-1 p24^gag. This conserved element vaccine is designed to avoid decoy epitopes by focusing responses to critical viral elements. We previously reported that vaccination of macaques with p24CE DNA induced robust cellular immune responses to CE that were not elicited upon wild type p55^gag DNA vaccination. p24CE DNA priming followed by p55^gag DNA boost provided a novel strategy to increase the magnitude and breadth of the cellular immune responses to HIV-1 Gag, including the induction of strong, multifunctional T-cell responses targeting epitopes within CE. Here, we examined the humoral responses induced upon p24CE DNA or p55^gag DNA vaccination in macaques and found that although both vaccines induced robust p24^gag binding antibody responses, the responses induced by p24CE DNA showed a unique broad range of linear epitope recognition. In contrast, antibodies elicited by p55^gag DNA vaccine failed to recognize p24CE protein and did not recognize linear epitopes spanning the CE. Interestingly, boosting of p24CE DNA primed animals with p55^gag DNA resulted in augmentation of antibodies able to recognize p24^gag as well as the p24CE proteins, thereby inducing broadest immunity. Our results indicate that an effectively directed vaccine strategy that includes priming with the conserved element vaccine followed by boost with the complete immunogen induces broad cellular and humoral immunity focused on the conserved regions of the virus. This novel and effective strategy to broaden responses could be applied against other antigens of highly diverse pathogens.     

Targeting human langerin promotes HIV-1 specific humoral immune responses

The main avenue for the development of an HIV-1 vaccine remains the induction of protective antibodies. A rationale approach is to target antigen to specific receptors on dendritic cells (DC) via fused monoclonal antibodies (mAb). In mouse and non-human primate models, targeting of skin Langerhans cells (LC) with anti-Langerin mAbs fused with HIV-1 Gag antigen drives antigen-specific humoral responses. The development of these immunization strategies in humans requires a better understanding of early immune events driven by human LC. We therefore produced anti-Langerin mAbs fused with the HIV-1 gp140z Envelope (αLC.Env). First, we show that primary skin human LC and in vitro differentiated LC induce differentiation and expansion of naïve CD4⁺ T cells into T follicular helper (Tfh) cells. Second, when human LC are pre-treated with αLC.Env, differentiated Tfh cells significantly promote the production of specific IgG by B cells. Strikingly, HIV-Env-specific Ig are secreted by HIV-specific memory B cells. Consistently, we found that receptors and cytokines involved in Tfh differentiation and B cell functions are upregulated by LC during their maturation and after targeting Langerin. Finally, we show that subcutaneous immunization of mice by αLC.Env induces germinal center (GC) reaction in draining lymph nodes with higher numbers of Tfh cells, Env-specific B cells, as well as specific IgG serum levels compared to mice immunized with the non-targeting Env antigen. Altogether, we provide evidence that human LC properly targeted may be licensed to efficiently induce Tfh cell and B cell responses in GC.